Genes that increase plant oil and method for using the same

ABSTRACT

This invention is intended to be used to search for a transcription factor having novel functions of increasing the weight of an individual plant, increasing the weight of a given tissue per individual plant, or improving the productivity of a given substance per individual plant and to improve such properties in the plant. The weight of an individual plant is increased, the weight of a given tissue per individual plant is increased, the productivity of a given substance per individual plant is improved, or the content of a given substance per given tissue of a plant is increased via expression of a transcription factor that has been modified to suppress transcription accelerating activity.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. patent application Ser. No.12/746,577, filed Sep. 8, 2010, which is a National Stage ofInternational Application No. PCT/JP2008/072159 filed Dec. 5, 2008,claiming priority based on Japanese Patent Application No. 2007-315267,filed Dec. 5, 2007, the contents of all of which are incorporated hereinby reference in their entirety.

BACKGROUND ART

The term “biomass” generally refers to the total amount of organismsthat inhabit or exist in a given area. When such term is used forplants, in particular, the term refers to dry weight per unit area. Abiomass unit is quantified in terms of a mass or an energy amount. Inthe case of plant biomass, the term “standing crop” is occasionally usedto represent “biomass.” Since plant biomass is generated by fixingatmospheric carbon dioxide with the use of the solar energy, it can beregarded as so-called “carbon-neutral energy.” Accordingly, an increaseof plant biomass is effective for global environmental preservation, theprevention of global warming, and mitigation of greenhouse gasemissions. Thus, technologies for increasing the production of plantbiomass have been industrially significant.

Plants are cultivated for the purpose of using some tissues thereof(e.g., seeds, roots, leaves, or stems) or for the purpose of producingvarious materials, such as a fat and oil. Examples of fat and oilproduced from plants that have been heretofore known include soybeanoil, sesame oil, olive oil, coconut oil, rice oil, cottonseed oil,sunflower oil, corn oil, safflower oil, and rapeseed oil. Such fat andoil are extensively used for household and industrial applications.Also, a fat and oil produced from plants is used as biodiesel fuels, andthe applicability thereof is increasing for alternative energy topetroleum.

Under such circumstances, it is necessary for the industrial success ofthe production of the fat and oil using plants that the productivity perunit of cultivation area be improved. If the number of cultivated plantsis assumed to be constant per unit of cultivation area, an improvementin the amount of fat and oil production per plant is found to benecessary. When fat and oil are extracted from seeds obtained fromplants, an improvement in the amount of fat and oil production per plantcan be achieved via techniques of, for example, improving the seed yieldper plant or increasing the fat and oil content in seeds.

Techniques for increasing the amount of fat and oil production fromplant seeds are roughly classified into techniques based on animprovement in cultivation methods and techniques based on thedevelopment of plant varieties that can increase the amount of fat andoil production. Techniques based on the development of plant varietiesare roughly classified as conventional breeding techniques such ascrossing and molecular breeding techniques via genetic recombination. Astechniques for increasing the amount of fat and oil production viagenetic recombination, A) a method of modifying synthetic pathways fortriacylglycerol (TAG) of seeds, which is a main component of plant fatand oil, and B) a method of modifying regulatory genes that regulateplant morphogenesis or metabolism are known.

In the method A) above, the amount of TAGs synthesized from sugarsproduced via photosynthesis can be increased by (1) enhancing synthesisactivities of a fatty acids (i.e., TAG components) or a glycerol fromsugars or (2) reinforcing the reaction of synthesizing TAGs fromglycerol and fatty acids. In this regard, the following techniques havebeen reported as techniques using genetically engineering techniques. Anexample of (1) is a technique in which cytosolic Acetyl-coenzyme Acarboxylase (ACCase) of Arabidopsis thaliana is overexpressed inplastids of Brassica rapa L. ver. Nippo-oleifera and the fat and oilcontent in seeds is improved by 5% (Plant Physiology, 1997, Vol. 113,pp. 75-81).

An example of (2) is a technique of increasing the fat and oilproduction via overexpression of diacylglycerol acyltransferase (DGAT)that transfers an acyl group to the sn-3 position of diacylglycerol(Plant Physiology, 2001, Vol. 126, pp. 861-874). It is reported that thefat and oil content and the seed weight are increased as the DGATexpression level increases, and the number of seeds per plant may beoccasionally increased according to the method of Plant Physiology,2001, Vol. 126, pp. 861-874. The fat and oil content in Arabidopsisthaliana seeds was increased by 46% and the fat and oil amount per plantwas increased by a maximum of about 125% by such technique.

As the method of B), expression of transcriptional factor genesassociated with regulation of biosynthetic enzyme genes expression maybe regulated. An example thereof is WO 01/35727. WO 01/35727 employs atechnique in which recombinant plants are prepared via exhaustiveoverexpression or knocking out of transcriptional factors and genes thatenhance the fat and oil content in seeds are then selected. WO 01/35727discloses that overexpression of ERF subfamily B-4 transcriptionalfactor genes results in a 23% increase in the fat and oil content inseeds. WO 01/35727, however, does not disclose an increase or decreasein fat and oil content per plant. Also, Plant J., 2004, 40, 575-585discloses the overexpression of WRINKLED1, which is a transcriptionalfactor having the AP2/EREB domain, improves the fat and oil content inseeds.

Although molecular breeding techniques as described above intended forthe improvement of various traits have been developed, techniques forimproving the yield involving increasing the weight of plant, increasinga given tissue, or improving the productivity of target substances havenot yet been put to practical use.

Further, targets of techniques for increasing the production of targetsubstances (fat and oil, in particular) via genetic recombination aredicotyledonous plants such as Arabidopsis thaliana and Brassica rapa L.ver. Nippo-oleifera. Techniques targeting monocotyledonous plants, suchas rice and maize, are not yet known.

This is considered to be due to the following reasons. That is, trulyexcellent genes have not yet been discovered and new recombinantvarieties that are found effective at the test phase cannot exhibiteffects as expected during the practical phase under a variety ofnatural environments. In order to overcome such problems, the discoveryof dramatically effective new genes and the development of genesexhibiting effects under practical environments, even if theeffectiveness thereof is equivalent to that of existing genes, arenecessary.

DISCLOSURE OF THE INVENTION Object to be Attained by the Invention

Under given circumstances, the present invention is intended to be usedto search for a transcription factor having new functions of increasingthe weight of an individual plant, increasing the weight of a giventissue per individual plant, improving the productivity of a givensubstance per individual plant, or increasing the content of a givensubstance in a given tissue of a plant and to provide a technique thatis capable of improving such features in a plant.

Means for Attaining the Object

The present inventors have conducted concentrated studies in order toattain the above object. As a result, they discovered that expression ofa transcription factor that is modified so as to suppress transcriptionaccelerating activity would lead to an increase in the weight of anindividual plant, an increase in the weight of a given tissue perindividual plant, an improvement in the productivity of a givensubstance per individual plant, or an increase in the content of a givensubstance in a given tissue of a plant. This has led to the completionof the present invention.

The plant according to the present invention attained increasedindividual plant weight, increased weight of a given tissue perindividual plant, improved productivity of a given substance perindividual plant, or increased content of a given substance in a giventissue of a plant via expression of a transcription factor withsuppressed transcription accelerating activity.

In the present invention, transcription factor that belongs to thetranscription factor family including a transcription factor comprisingthe amino acid sequence as shown in SEQ ID NO: 2, a transcription factorcomprising the amino acid sequence as shown in SEQ ID NO: 4, atranscription factor comprising the amino acid sequence as shown in SEQID NO: 6, a transcription factor comprising the amino acid sequence asshown in SEQ ID NO: 8, a transcription factor comprising the amino acidsequence as shown in SEQ ID NO: 10, a transcription factor comprisingthe amino acid sequence as shown in SEQ ID NO: 12, and a transcriptionfactor comprising the amino acid sequence as shown in SEQ ID NO: 14 canbe used as the above-mentioned transcription factor.

The transcription factor is preferably any of proteins (a) to (c) below:

(a) a protein comprising the amino acid sequence as shown in SEQ ID NO:2, 4, 6, 8, 10, 12, or 14;

(b) a protein comprising an amino acid sequence derived from the aminoacid sequence as shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, or 14 bydeletion, substitution, addition, or insertion of 1 or a plurality ofamino acids and having transcription accelerating activity; or

(c) a protein encoded by a polynucleotide hybridizing under stringentconditions to a polynucleotide comprising a nucleotide sequencecomplementary to the nucleotide sequence as shown in SEQ ID NO: 1, 3, 5,7, 9, 11, or 13 and having transcription accelerating activity.

In particular, the plant according to the present invention can havesuppressed transcription accelerating activity of a target transcriptionfactor by expressing a chimeric protein resulting from the fusion of thetarget transcription factor with a functional peptide that converts anarbitrary transcription factor into a transcription repressor in aplant. Examples of the functional peptides include peptides representedby formulae (1) to (8) below:

(1) X1-Leu-Asp-Leu-X2-Leu-X3

wherein X1 represents 0 to 10 amino acid residues; X2 represents Asn orGlu; and X3 represents at least 6 amino acid residues;

(2) Y1-Phe-Asp-Leu-Asn-Y2-Y3

wherein Y1 represents 0 to 10 amino acid residues; Y2 represents Phe orIle; and Y3 represents at least 6 amino acid residues;

(3) Z1-Asp-Leu-Z2-Leu-Arg-Leu-Z3

wherein Z1 represents Leu, Asp-Leu, or Leu-Asp-Leu; Z2 represents Glu,Gln, or Asp; and Z3 represents 0 to 10 amino acid residues;

(4) Asp-Leu-Z4-Leu-Arg-Leu

wherein Z4 represents Glu, Gln, or Asp;

(5) α1-Leu-β1-Leu-γ1-Leu;

(6) α1-Leu-β1-Leu-γ2-Leu;

(7) α1-Leu-β2-Leu-Arg-Leu; and

(8) α2-Leu-β1-Leu-Arg-Leu;

wherein, in formulae (5) to (8), α1 represents Asp, Asn, Glu, Gln, Thr,or Ser; α2 represents Asn, Glu, Gln, Thr, or Ser; β1 represents Asp,Gln, Asn, Arg, Glu, Thr, Ser, or His; β2 represents Asn, Arg, Thr, Ser,or His; γ1 represents Arg, Gln, Asn, Thr, Ser, His, Lys, or Asp; and γ2represents Gln, Asn, Thr, Ser, His, Lys, or Asp.

In the plant according to the present invention, the seed weight as theweight of a given tissue can be improved. Also, in the plant accordingto the present invention, the productivity of a fat and oil as theproductivity of a given substance described above can be improved.

The present invention can provide a method for producing a plantexhibiting increased individual plant weight, increased weight of agiven tissue per individual plant, improved productivity of a givensubstance per individual plant, or increased content of a givensubstance in a given tissue of a plant via expression of a transcriptionfactor with suppressed transcription accelerating activity.

Further, the present invention can provide a chimeric protein resultingfrom the fusion of a target transcription factor with a functionalpeptide that converts an arbitrary transcription factor into atranscription repressor, which is capable of increasing the weight of anindividual plant, increasing the weight of a given tissue per individualplant, improving the productivity of a given substance per individualplant, or increasing the content of a given substance in a given tissueof a plant via suppression of transcription accelerating activity of thetranscription factor; a polynucleotide encoding the chimeric protein; arecombinant expression vector containing the polynucleotide and apromoter; and a kit for improving properties of a plant in terms of theweight of a plant, the weight of a given tissue, the productivity of agiven substance, or the content of a substance comprising the expressionvector.

Effects of the Invention

The plant according to the present invention exhibits increasedindividual plant weight, increased weight of a given tissue perindividual plant, improved productivity of a given substance perindividual plant, or increased content of a given substance in a giventissue of a plant, compared with a wild-type plant. With the use of theplant according to the present invention, accordingly, the amount ofproduction of the target biomass can be increased, the yield of thetarget tissue can be increased, the productivity of the target substancecan be improved, and the content of the target substance in the targettissue can be increased. This enables production of biomass, planttissue, or target substances at low cost.

Also, the chimeric protein according to the present invention can imparta plant with traits such as increased individual plant weight, increasedweight of a given tissue per individual plant, improved productivity ofa given substance per individual plant, or increased content of a givensubstance in a given tissue of a plant, compared with a wild-type plant.With the use of the chimeric protein according to the present invention,accordingly, a plant that can realize an increased amount of biomassproduction, increased yield of the target tissue, improved productivityof a target substance, or increased content of a target substance in thetarget tissue can be produced.

This description includes part or all of the contents as disclosed inthe description and/or drawings of Japanese Patent Application No.2007-315267, which is a priority document of the present application.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a characteristic diagram showing the results of measuring fatand oil contents in seeds of plants prepared in the examples (T2plant-T3 seeds).

FIG. 2 is a characteristic diagram showing the results of measuring theseed yields of plants prepared in the examples (T2 plant-T3 seeds).

FIG. 3 is a characteristic diagram showing the results of calculatingthe amount of fat and oil production per individual plant of plantsprepared in the examples (T2 plant-T3 seeds).

FIG. 4 is a characteristic diagram showing the results of measuring theamount of biomass of plants prepared in the examples (T2 plant-T3seeds).

BEST MODES FOR CARRYING OUT THE INVENTION

Hereafter, the present invention is described in detail.

The plant according to the present invention exhibits increasedindividual plant weight, increased weight of a given tissue perindividual plant, improved productivity of a given substance perindividual plant, or increased content of a given substance in a giventissue, compared with a wild-type plant, via expression of atranscription factor with suppressed transcription acceleratingactivity. Specifically, the plant according to the present invention wasproduced by expressing a transcription factor with suppressedtranscription accelerating activity in a plant of interest, so as tosignificantly improve the weight of a plant, the weight of a giventissue, the productivity of a given substance, or the content of a givensubstance therein.

The term “the increased weight of a plant” used herein refers to anincrease in production of so-called biomass, i.e., an increase in theamount of biomass per given area. The amount of biomass produced pergiven area can be increased by increasing the planting density (i.e.,the number of individual plants per given area) and by increasing theweight or energy amount per individual plant. Specifically, plantbiomass can be evaluated in terms of dry weight per individual plant, aswell as in terms of dry weight per given area.

In the present invention, accordingly, biomass may be defined in termsof the plant dry weight per individual plant, the dry weight of aerialparts per individual plant, the weight of a given tissue accumulatingthe target product per individual plant, the target product perindividual plant, or the content of the target substance per giventissue.

The term “the weight of a given tissue per individual plant” used hereinrefers to the weight of at least 1 tissue selected from among tissuessuch as seeds, roots, leaves, stems, flowers, and pollen that constituteplants. Particularly preferably, the plant according to the presentinvention is intended to increase seed weight.

The term “the productivity of a given substance per individual plant”used herein refers to the contents of various substances generated byplants per individual plant. Substances are not particularly limited andmay be naturally produced by plants. Alternatively, such substances maybe not naturally produced by plants, but rather may be produced fromplants via genetic engineering or other means. If the content of thetarget product per tissue is increased, in particular, purification andtransportation costs can be reduced, and the industrial usefulness ofsuch plants is significant. Specifically, target products may belignocelluloses that account for substantially the entire weight of aplant, plant fat and oil that is used as seed oils at the industriallevel may be preferably used, and plant oils are particularlypreferable. Plant oils may be simple lipids that is the esters of fattyacids with alcohols, complex lipid including phosphorus, sugar,nitrogen, and the like, or a fatty acid. An alcohol of a simple lipidmay be a higher alcohol having a high molecular weight or a polyhydricalcohol, such as glycerol (glycerin). A fatty acid of a simple lipid maybe a saturated fatty acid, unsaturated fatty acid, or special fatty acidcomprising a hydroxyl group or an epoxy group. Simple lipids that arethe esters of glycerol and fatty acid may be monoacylglycerol,diacylglycerol, or triacylglycerol.

Hereafter, substances that improve productivity are described withreference to a fat and oil, although the technical scope of the presentinvention is not limited thereto. The present invention is alsoapplicable to substances other than the fat and oil as substancesgenerated from plants.

The present invention can cover any plants without particularlimitation. Angiosperms are particularly preferable as plants, andeither monocotyledonous or dicotyledonous plants may be covered. Plantsthat have been heretofore used for the production of the fat and oil areparticularly preferable. Examples of intended plants include soybeans,sesame, olive oils, coconuts, rice, cottons, sunflowers, maize,safflowers, and rapeseeds. Also, Arabidopsis thaliana, which isextensively used as a model organism in genetic analysis of plants andfor which a method for gene expression analysis has been established canbe intended.

The term “transcription factor with suppressed transcriptionaccelerating activity” refers to a transcription factor havingtranscription accelerating activity significantly lower than theactivity that the transcription factor would naturally have. Methods forlowering transcription accelerating activity are not particularlylimited. Gene-silencing techniques can be extensively employed, and amethod of constructing a fusion protein to which a repressor domainsequence has been added is the most preferable.

In such a technique, “repressor domain sequences” are amino acidsequences constituting peptides that convert arbitrary transcriptionfactors into transcription repressors, and the present inventors havediscovered a wide variety of such sequences.

Techniques involving the use of repressor domain sequences are disclosedin, for example, JP Patent Publication (kokai) No. 2001-269177 A, JPPatent Publication (kokai) No. 2001-269178 A, JP Patent Publication(kokai) No. 2001-292776 A, JP Patent Publication (kokai) No. 2001-292777A, JP Patent Publication (kokai) No. 2001-269176 A, JP PatentPublication (kokai) No. 2001-269179 A, WO 03/055903, Ohta, M., Matsui,K., Hiratsu, K., Shinshi, H. and Ohme-Takagi, M., The Plant Cell, Vol.13, 1959-1968, August, 2001, and Hiratsu, K., Ohta, M., Matsui, K.,Ohme-Takagi, M., FEBS Letters 514, 2002, 351-354. Repressor domainsequences are cleaved from Class II ethylene-responsive element bindingfactor (ERF) proteins or plant zinc finger proteins (e.g., theArabidopsis thaliana SUPERMAN protein) and have very simple structures.

Examples of transcription factors with transcription acceleratingactivity to be suppressed include the transcription factor identified asAt3g15510 in Arabidopsis thaliana (hereafter simply referred to as the“transcription factor At3g15510”), the transcription factor identifiedas At5g24520 in Arabidopsis thaliana (hereafter simply referred to asthe “transcription factor At5g24520”), the transcription factoridentified as At5g07580 in Arabidopsis thaliana (hereafter simplyreferred to as the “transcription factor At5g07580”), the transcriptionfactor identified as At1g74930 in Arabidopsis thaliana (hereafter simplyreferred to as the “transcription factor At1g74930”), the transcriptionfactor identified as At5g47390 in Arabidopsis thaliana (hereafter simplyreferred to as the “transcription factor At5g47390”), the transcriptionfactor identified as At5g25190 in Arabidopsis thaliana (hereafter simplyreferred to as the “transcription factor At5g25190”), and thetranscription factor identified as At3g61910 in Arabidopsis thaliana(hereafter simply referred to as the “transcription factor At3g61910”).

There is no report regarding functions of the transcription factorAt3g15510. The transcription factor At5g24520 is a transcription factorhaving the WD40 repeat, it is known as a TTG1 gene, and functionsthereof that regulates the flavonoid/anthocyanin synthesis (Plant Cell,2001 September; 13 (9): 2099-114, and Plant J. 2006 June; 46 (5):768-79) or patterning of epidermal cells (e.g., trichome or root hair)(Curr Opin Plant Biol., 2003 February; 6 (1): 74-8) have been reported.The transcription factor At5g07580 is classified into the B-3 subfamilyof the AP2/ERF family, and there is no report regarding functionsthereof. The transcription factor At1g74930 is classified into the A-5subfamily of the AP2/ERF family, and there is no report regardingfunctions thereof. The transcription factor At5g47390 is a transcriptionfactor of the myb family protein, and there is no report regardingfunctions thereof. The transcription factor At5g25190 is a transcriptionfactor of the AP2/ERF family, and there is no report regarding functionsthereof. The transcription factor At3g61910 is an NAC transcriptionfactor. The transcription factor At3g61910 is reported as atranscription factor that regulates secondary thickening of a cell wall(Plant Cell, 2005 November; 17 (11): 2993-3006). Also, it is reportedthat overexpression of genes of the transcription factor At3g61910 towhich a repressor domain sequence had been added suppresses secondarythickening of a cell wall.

The amino acid sequences of such transcription factors and thenucleotide sequences of the coding regions of the genes encoding suchtranscription factors are summarized in Table 1.

TABLE 1 Transcription factor Amino acid sequence Nucleotide SequenceAt3g15510 SEQ ID NO: 2 SEQ ID NO: 1 At5g24520 SEQ ID NO: 4 SEQ ID NO: 3At5g07580 SEQ ID NO: 6 SEQ ID NO: 5 At1g74930 SEQ ID NO: 8 SEQ ID NO: 7At5g47390 SEQ ID NO: 10 SEQ ID NO: 9 At5g25190 SEQ ID NO: 12 SEQ ID NO:11 At3g61910 SEQ ID NO: 14 SEQ ID NO: 13

The specific transcription factors with transcription acceleratingactivity to be suppressed are not limited to those comprising the aminoacid sequences as shown in SEQ ID NOs: 2, 4, 6, 8, 10, 12, and 14. Anintended transcription factor may be a transcription factor comprisingan amino acid sequence derived from the amino acid sequence as shown inSEQ ID NO: 2, 4, 6, 8, 10, 12, or 14 by deletion, substitution,addition, or insertion of 1 or a plurality of amino acids and havingtranscription accelerating activity. The number of such plurality ofamino acids is, for example, 1 to 20, preferably 1 to 10, morepreferably 1 to 7, further preferably 1 to 5, and particularlypreferably 1 to 3. Deletion, substitution, or addition of amino acidscan be conducted by modifying a nucleotide sequence encoding theabove-mentioned transcription factor via a method known in the art.Mutation can be introduced into a nucleotide sequence via known methods,such as the Kunkel or Gapped duplex method, or methods in accordancetherewith. For example, mutation is introduced with the use ofmutagenesis kits utilizing site-directed mutagenesis (e.g., Mutant-K orMutant-G (tradenames, manufactured by TAKARA)) or the LA PCR in vitroMutagenesis Series Kit (tradename, manufactured by TAKARA).

Further, transcription factors with transcription accelerating activityto be suppressed are not limited to transcription factors At3g15510,At5g24520, At5g07580, At1g74930, At5g47390, At5g25190, and At3g61910 inArabidopsis thaliana, and transcription factors (hereafter referred toas “homologous transcription factors) having equivalent functions inplants other than Arabidopsis thaliana (e.g., plants mentioned above)are within the scope of the present invention. The homologoustranscription factors corresponding to the transcription factorsAt3g15510, At5g24520, At5g07580, At1g74930, At5g47390, At5g25190, andAt3g61910 can be searched for, in case that the plant genome informationhas been revealed, using the genome information of the intended plantbased on the amino acid sequences of the transcription factorsAt3g15510, At5g24520, At5g07580, At1g74930, At5g47390, At5g25190, andAt3g61910 or the nucleotide sequences of the genes encoding suchtranscription factors. As a homologous transcription factor, an aminoacid sequence having, for example, 70% or higher, preferably 80% orhigher, more preferably 90% or higher, and most preferably 95% or higherhomology to the amino acid sequence of any of the above transcriptionfactors is searched for. Homology values are determined by default usinga computer program that implements the BLAST algorithm and a databasethat stores gene sequence information.

In case that the genome information of intended plants has not beenrevealed, the genome is extracted from the intended plant, or a cDNAlibrary of the intended plant is constructed. The genome region or cDNAhybridizing under stringent conditions to at least part of thenucleotide sequence of the gene of transcription factor At3g15510,At5g24520, At5g07580, At1g74930, At5g47390, At5g25190, or At3g61910 isthen isolated. Thus, a homologous gene can be identified. Understringent conditions, hybridization is carried out via washing at 60° C.in the presence of 2×SSC while maintaining a bond. Hybridization can becarried out in accordance with a conventional technique, such as themethod disclosed by J. Sambrook et al. Molecular Cloning, A LaboratoryManual, 2nd Ed., Cold Spring Harbor Laboratory, 1989.

The plant according to the present invention significantly improves theamount of fat and oil production via expression of the above-describedtranscription factor with suppressed transcription acceleratingactivity. In such plant, the endogenous transcription factor may bemodified and transcription accelerating activity thereof may besuppressed. Alternatively, a gene encoding a modified transcriptionfactor with suppressed transcription accelerating activity may beintroduced and such gene may be expressed. Transcription acceleratingactivity of the gene encoding the target transcription factor may besuppressed via a so-called gene-silencing technique.

A preferable example of such technique is a technique comprisingintroducing a gene encoding a fusion protein resulting from the fusionof the aforementioned transcription factor with a functional peptidethat converts an arbitrary transcription factor into a transcriptionrepressor into an intended plant and expressing such fusion proteintherein.

A functional peptide that converts an arbitrary transcription factorinto a transcription repressor (hereafter referred to as a“transcription repressor converting peptide”) used herein is notparticularly limited, as long as it can form a chimeric protein fusedwith the transcription factor, thereby suppressing transcription of thetarget gene regulated by the transcription factor. Such transcriptionrepressor converting peptide is described in detail in JP PatentPublication (kokai) No. 2005-204657 A, and all peptides disclosedtherein can be used.

Examples of transcription repressor converting peptides include aminoacid sequences represented by formulae (1) to (8) below:

(1) X1-Leu-Asp-Leu-X2-Leu-X3

wherein X1 represents 0 to 10 amino acid residues; X2 represents Asn orGlu; and X3 represents at least 6 amino acid residues;

(2) Y1-Phe-Asp-Leu-Asn-Y2-Y3

wherein Y1 represents 0 to 10 amino acid residues; Y2 represents Phe orIle; and Y3 represents at least 6 amino acid residues;

(3) Z1-Asp-Leu-Z2-Leu-Arg-Leu-Z3

wherein Z1 represents Leu, Asp-Leu, or Leu-Asp-Leu; Z2 represents Glu,Gln, or Asp; and Z3 represents 0 to 10 amino acid residues;

(4) Asp-Leu-Z4-Leu-Arg-Leu

wherein Z4 represents Glu, Gln, or Asp;

(5) α1-Leu-β1-Leu-γ1-Leu;

(6) α1-Leu-β1-Leu-γ2-Leu;

(7) α1-Leu-β2-Leu-Arg-Leu; and

(8) α2-Leu-β1-Leu-Arg-Leu

wherein, in formulae (5) to (8), α1 represents Asp, Asn, Glu, Gln, Thr,or Ser; α2 represents Asn, Glu, Gln, Thr, or Ser; β1 represents Asp,Gln, Asn, Arg, Glu, Thr, Ser, or His; β2 represents Asn, Arg, Thr, Ser,or His; γ1 represents Arg, Gln, Asn, Thr, Ser, His, Lys, or Asp; and γ2represents Gln, Asn, Thr, Ser, His, Lys, or Asp.Transcription Repressor Converting Peptide Represented by Formula (1)

The number of amino acid residues represented by X1 of the transcriptionrepressor converting peptide represented by formula (1) may be 0 to 10.Specific types of amino acids that constitute the amino acid residuesrepresented by X1 are not particularly limited, and any amino acid maybe used. It is preferable that the number of amino acid residuesrepresented by X1 be as small as possible from the viewpoint of ease ofsynthesis of the transcription repressor converting peptide representedby formula (1). Specifically, the number of amino acid residuesrepresented by X1 is preferably 5 or less.

Also, the number of the amino acid residues represented by X3 of thetranscription repressor converting peptide represented by formula (1)may be at least 6. Specific types of amino acids that constitute theamino acid residues represented by X3 are not particularly limited, andany amino acid may be used.

Transcription Repressor Converting Peptide Represented by Formula (2)

The number of the amino acid residues represented by Y1 of thetranscription repressor converting peptide represented by formula (2)may be 0 to 10 as in the case of X1 of the transcription repressorconverting peptide represented by formula (1). Also, specific types ofamino acids that constitute the amino acid residues represented by Y1are not particularly limited, and any amino acid may be used.Specifically, the number of amino acid residues represented by Y1 ispreferably 5 or less.

Also, the number of the amino acid residues represented by Y3 of thetranscription repressor converting peptide represented by formula (2)may be at least 6, as in the case of X3 of the transcription repressorconverting peptide represented by formula (1). Also, specific types ofamino acids that constitute the amino acid residues represented by Y3are not particularly limited, and any amino acid may be used.

Transcription Repressor Converting Peptide Represented by Formula (3)

The amino acid residues represented by Z1 of the transcription repressorconverting peptide represented by formula (3) comprise 1 to 3 Leuresidues: i.e., Leu when the number of amino acids is 1; Asp-Leu whenthe number of amino acids is 2; and Leu-Asp-Leu when the number of aminoacids is 3.

In contrast, the number of the amino acid residues represented by Z3 ofthe transcription repressor converting peptide represented by formula(3) may be 0 to 10. Also, specific types of amino acids that constitutethe amino acid residues represented by Z3 are not particularly limited,and any amino acid may be used. Specifically, the number of amino acidresidues represented by Z3 is more preferably 5 or less. Specificexamples of amino acid residues represented by Z3 include, but are notlimited to, Gly, Gly-Phe-Phe, Gly-Phe-Ala, Gly-Tyr-Tyr, and Ala-Ala-Ala.

The number of amino acid residues constituting the entire transcriptionrepressor converting peptide represented by formula (3) is notparticularly limited. From the viewpoint of ease of synthesis, thenumber of amino acids is preferably 20 or less.

Transcription Repressor Converting Peptide Represented by Formula (4)

The transcription repressor converting peptide represented by formula(4) is a hexamer (6-mer) comprising 6 amino acid residues. When theamino acid residue represented by Z4 of the transcription repressorconverting peptide represented by formula (4) is Glue, the amino acidsequence of interest is equivalent to the amino acid sequence composedof amino acids 196 to 201 of the Arabidopsis thaliana SUPERMAN protein(SUP protein).

Various transcription repressor converting peptides described above canfuse to the above-described transcription factors to result in fusionproteins, and such peptides can convert the transcription factors intotranscription repressors. According to the present invention, therefore,fusion proteins can be produced using polynucleotides encoding thetranscription repressor converting peptides to obtain fusion genesthereof with genes encoding the transcription factors.

More specifically, polynucleotides encoding the transcription repressorconverting peptides (hereafter referred to as the “transcriptionrepressor converting polynucleotides”) are ligated to the genes encodingthe transcription factors to construct fusion genes, and the resultingfusion genes are introduced into plant cells. Thus, fusion proteins canbe produced. Specific nucleotide sequences of the transcriptionrepressor converting polynucleotides are not particularly limited, andsuch polynucleotides may comprise nucleotide sequences corresponding tothe amino acid sequences of the transcription repressor convertingpeptides based on genetic codes. The transcription repressor convertingpolynucleotides may comprise nucleotide sequences that serve as ligationsites to be connected to the transcription factor genes, as necessary.When the amino acid reading frame of the transcription repressorconverting polynucleotide is not aligned with that of the transcriptionfactor gene, the polynucleotide may further comprise an additionalnucleotide sequence, so as to align the reading frames. Further, thepolynucleotide may comprise various additional polypeptides, such as apolypeptide having a linker function for connecting the transcriptionfactor to the transcription repressor converting peptide or apolypeptide for labeling a fusion protein with an epitope, such as His,Myc, or Flag. Further, the fusion protein may comprise a structure otherthan a polypeptide, such as a sugar chain or an isoprenoid group,according to need.

The method for producing the plant according to the present invention isnot particularly limited, provided that the method comprises a step ofproducing a transcription factor with suppressed transcriptionaccelerating activity in a plant to improve the productivity of a fatand oil. An example thereof is a production method comprising steps ofconstruction of an expression vector, transformation, and selection.Such steps are described in detail below.

Step of Constructing Expression Vector

A step of constructing an expression vector is not particularly limited,provided that a recombinant expression vector comprising the geneencoding the above-mentioned transcription factor, the transcriptionrepressor converting polynucleotide, and a promoter is constructed. Avariety of known vectors can be used as bases for recombinant expressionvectors. Examples of vectors that can be used include plasmid, phage,and cosmid vectors, and adequate vectors can be selected in accordancewith the plant cells to which such vectors are introduced or methods ofintroduction into a cell. Specific examples include pBR322, pBR325,pUC19, pUC119, pBluescript, pBluescriptSK, and pBI vectors. When avector is introduced into plant by the Agrobacterium method, inparticular, use of the pBI binary vector is preferable. Specificexamples of pBI binary vectors include pBIG, pBIN19, pBI101, pBI121, andpBI221 vectors.

Promoters are not particularly limited, provided that such promoters canexpress a gene of interest in a plant. Known promoters can be preferablyused. Examples of such promoters include cauliflower mosaic virus 35Spromoters (CaMV 35S), actin promoters, ubiquitin promoters, noparinsynthase promoters, tobacco PR1a gene promoters, andribulose-1,5-bisphosphate carboxylase/oxygenase small subunit promotersin tomatoes. Among such promoters, cauliflower mosaic virus 35Spromoters, actin promoters, and ubiquitin promoters are preferable. Withthe use of such promoters, arbitrary genes can be intensively expressedupon introduction of the resulting recombinant expression vector intoplant cells. A promoter is ligated so as to express the fusion gene ofthe gene encoding the transcription factor with the transcriptionrepressor converting polynucleotide, and the resultant may be introducedinto the vector in that state. The specific structure of a recombinantexpression vector is not particularly limited.

The recombinant expression vector may further comprise other DNAsegments, in addition to the promoter and the fusion gene. Such otherDNA segments are not particularly limited, and examples thereof includea terminator, a selection marker, an enhancer, and a nucleotide sequencefor enhancing translation efficiency. Also, the recombinant expressionvector may further comprise a T-DNA region. The T-DNA region can enhancethe efficiency of gene introduction, particularly when introducing therecombinant expression vector into a plant with the use ofAgrobacterium.

A terminator is not particularly limited, provided that it functions asa transcription termination site, and a known terminator may be used.Specific examples of terminators that can be preferably used include thetranscription termination region of the noparin synthase gene (the Nosterminator) and the transcription termination region of the cauliflowermosaic virus 35S (the CaMV 35S terminator), with the Nos terminatorbeing preferable. The recombinant vector can be used to avoid theoccurrence of phenomena such as synthesis of an unnecessarily longtranscript after the introduction thereof into plant cells or areduction in the plasmid copy number caused by a potent promoter bypositioning a terminator in an adequate site.

Drug-resistance genes can be used as selection markers, for example.Specific examples of such drug-resistance genes include drug-resistancegenes that are resistant to hygromycin, bleomycin, kanamycin,gentamicin, and chloramphenicol. Plants that grow in a medium containingthe above antibiotics may be selected with the use of such selectionmarkers, so that transformed plants can be easily selected.

An example of a nucleotide sequence for enhancing translation efficiencyis the omega sequence derived from the tobacco mosaic virus. This omegasequence may be located in the untranslational region (5′ UTR) of thepromoter to enhance the translation efficiency of the fusion gene. Thus,the recombinant expression vector can comprise a variety of DNA segmentsin accordance with its intended purposes.

Methods for constructing recombinant expression vectors are notparticularly limited. The promoter, the gene encoding the transcriptionfactor, the transcription repressor converting polynucleotide, and,according to need, other DNA segments may be introduced into anadequately selected matrix vector in a predetermined order. For example,the gene encoding the transcription factor may be ligated to thetranscription repressor converting polynucleotide to construct a fusiongene, the fusion gene may then be ligated to the promoter (e.g., aterminator according to need) to construct an expression cassette, andthe resulting expression cassette may be introduced into the vector.

When constructing a fusion gene and an expression cassette, for example,cleavage sites of DNA segments are made to be protruding ends that arecomplementary to each other, such DNA segments are subjected to thereaction with the aid of ligation enzymes, and the order of such DNAsegments can be determined. When an expression cassette comprises aterminator, the expression cassette may comprise the promoter, thechimeric gene, and the terminator, in that order from upstream. Also,the types of reagents used for constructing a recombinant expressionvector (i.e., restriction enzymes or ligation enzymes) are notparticularly limited, and commercially available products may beadequately selected and used.

Also, methods for growing the recombinant expression vector (i.e.,methods of production) are not particularly limited, and known methodscan be employed. In general, E. coli hosts may be used, and therecombinant expression vector may be grown therein. In such a case,preferable E. coli species may be selected in accordance with a vectortype.

Step of Transformation

The step of transformation that is carried out in the present inventioncomprises introducing the recombinant expression vector into a plantcell in order to express the aforementioned fusion genes. Methods ofintroducing a recombinant expression vector into a plant cell (i.e.,methods of transformation) are not particularly limited, and adequateknown methods can be employed in accordance with a given plant cell.Specific examples of such methods include a method involving the use ofAgrobacterium and a method involving direct introduction of arecombinant expression vector into a plant cell. Examples of methodsinvolving the use of Agrobacterium that can be employed include methodsdescribed in Bechtold, E., Ellis, J., and Pelletier, G., 1993, In PlantaAgrobacterium-mediated gene transfer by infiltration of adultArabidopsis plants, C. R. Acad. Sci. Paris Sci. Vie, 316, 1194-1199 andZyprian E., Kado C. L., Agrobacterium-mediated plant transformation bynovel mini-T vectors in conjunction with a high-copy vir region helperplasmid, Plant Molecular Biology, 1990, 15 (2), 245-256.

Examples of methods involving direct introduction of a recombinantexpression vector into a plant cell include microinjection,electroporation, the polyethylene glycol method, the particle gunmethod, the protoplast fusion method, and the calcium phosphate method.

Examples of plant cells into which the recombinant expression vector isto be introduced include tissue cells in plant organs such as flowers,leaves, and roots, calluses, and suspension cultured cells. According tothe method for producing plants according to the present invention, therecombinant expression vector may be adequately constructed inaccordance with the type of plant to be produced. Alternatively, ageneral-purpose recombinant expression vector may be constructed inadvance and it may be introduced into a plant cell. Specifically, themethod for producing plants according to the present invention may ormay not comprise the step of constructing the recombinant expressionvector.

Other Steps and Other Methods

The method for producing the plant according to the present inventionmay comprise a method of transformation. Further, the method maycomprise a method for constructing a recombinant expression vector andother steps. Specifically, the method may comprise a step of selectingadequate transformants from transformed plants.

Methods of selection are not particularly limited. For example,transformants may be selected based on, for example, drug resistance,such as hygromycin-resistance, or based on the content of fat and oil inplants or arbitrary organs or tissues after the transformed plants havebeen grown. For example, transformants may be selected based on fat andoil content by quantifying the fat and oil components in seeds of thetransformants in accordance with a conventional technique and comparingthe quantified value with the fat and oil content in seeds ofnon-transformed plants (see the examples below).

According to the method for producing the plant according to the presentinvention, the fusion gene is introduced into a plant. Thus, offspringplants exhibiting significantly improved fat and oil content can beobtained from such plant via sexual or asexual reproduction. Also, plantcells or reproductive materials, such as seeds, fruits, stocks,calluses, tubers, cut ears, or lumps, may be obtained from a plant or anoffspring plant thereof, and a plant of interest can be mass-producedtherefrom. The method for producing the plant according to the presentinvention, accordingly, may comprise a step of growing the selectedplant (i.e., the step of mass production).

The term “plant” used herein refers to a grown plant, a plant cell, aplant tissue, a callus, or a seed. According to the present invention,specifically, substances that can eventually grow into individual plantsare regarded as plants. Plant cells can exist in various forms. Examplesof such plant cells include suspension cultured cells, protoplasts, andleaf sections. Such plant cells may be grown and differentiated toobtain plants. Plants can be reproduced from plant cells via a knowntechnique in accordance with plant cell type. The method for producingthe plant according to the present invention, accordingly, may comprisea step of reproducing plants from plant cells or the like.

The method for producing the plant according to the present invention isnot limited to a method in which transformation is carried out with theaid of a recombinant expression vector, and other methods may beemployed. Specifically, a fusion protein may be introduced into a plant,for example. In such a case, a fusion protein may be introduced into ayoung plant so as to improve the fat and oil content in a site of aplant that is to be eventually used. Methods for introducing a fusionprotein are not particularly limited, and various known methods may beemployed.

As described above, the present invention can provide a plant into whicha transcription factor with suppressed transcription acceleratingactivity has been introduced and in which fat and oil content has beensignificantly improved. A transcription factor having transcriptionaccelerating activity is also expressed in the plant according to thepresent invention; however, the transcription factor with suppressedtranscription accelerating activity can suppress gene expression in adominant-negative manner. This varies the expression levels of genesinvolved in fat and oil production and/or genes involved indecomposition of the produced fat and oil in the plant according to thepresent invention. This can result in the significantly enhanced fat andoil content.

The condition of “significantly enhanced fat and oil content” refers toa situation in which fat and oil content has been enhanced, althoughseed mass per grain has not changed compared with wild-type plants, or asituation in which fat and oil content has been enhanced withsignificantly increased seed mass per grain compared with wild-typeplants. Both cases indicate increased amounts of fat and oil produced byan individual plant. The plant according to the present invention can beused for the method for producing plant-derived fat and oil. Forexample, the plant according to the present invention is allowed togrow, seeds are collected, and fat and oil components are extracted fromthe collected seeds. Thus, the fat and oil can be produced.

It can be said that the method for producing fat and oil utilizing theplant according to the present invention is excellent particularly interms of productivity because of the high fat and oil content in anindividual plant. If the number of cultivated plants is assumed to beconstant per unit of cultivation area, specifically, the amount of fatand oil produced per unit of cultivation area is significantly increasedwith the use of the plant according to the present invention. With theuse of the plant according to the present invention, accordingly,production costs required for the production of fat and oil can beremarkably reduced.

In the method for producing fat and oil using the plant according to thepresent invention, the fat and oil to be produced are not particularlylimited. Examples thereof include plant-derived fat and oil, such assoybean oil, sesame oil, olive oil, coconut oil, rice oil, cottonseedoil, sunflower oil, corn oil, safflower oil, and rapeseed oil. Theproduced fat and oil can be extensively used for household or industrialapplications. Further, such fat and oil can be used as startingmaterials for biodiesel fuels. With the use of the plant according tothe present invention, specifically, such fat and oil for household orindustrial applications, biodiesel fuels, and the like can be producedat low cost. An improved seed yield per plant can result in animprovement in the productivity of feeds and food products, in additionto the productivity of fat and oil, and production costs can be reduced.Also, an increased amount of biomass per plant can result in animprovement in the productivity of biomass after seed harvesting or theentire biomass. Biomass can be adequately treated to be degraded intosugar. Sugar can be converted into a variety of chemical substances,including ethanol, by a fermentation method utilizing microorganisms.Also, biomass may be directly combusted to obtain thermal energy or anelectric energy may be obtained from the thermal energy. With the use ofthe plant provided by the present invention, chemical substances,thermal energy, electric energy, and the like described above can beproduced in a cost-effective manner.

EXAMPLES

Hereafter, the present invention is described in greater detail withreference to the examples, although the technical scope of the presentinvention is not limited to the examples.

Example 1

In this example, fusion proteins of Arabidopsis thaliana transcriptionfactors At3g15510, At5g24520, At5g07580, At1g74930, At5g47390,At5g25190, and At3g61910 to which repressor domain sequences had beenadded were expressed in plants, and the fat and oil content of the seedsobtained from the plants was measured.

Amplification of Transcription Factor Genes

The genes encoding the above-mentioned transcription factors wereobtained from the Arabidopsis thaliana cDNA library, and the regionsexcluding the termination codons of such genes were amplified via PCRusing the primers shown below. PCR was carried out via denaturation at94° C. for 1 minute, annealing at 47° C. for 2 minutes, and elongationat 74° C. for 1 minute, and this cycle was repeated 25 times. After thecompletion of PCR, the amplified DNA fragment was separated via agarosegel electrophoresis and recovered.

Forward primer for amplifying At3g15510 (SEQ ID NO: 15)GATGGAGAGCACCGATTCTTCCGGTGGTCC  Reverse primer for amplifying At3g15510(SEQ ID NO: 16) AGAAGAGTACCAATTTAAACCGGGTAATT Forward primer for amplifying At5g24520 (SEQ ID NO: 17)GATGGATAATTCAGCTCCAGATTCGTTATC  Reverse primer for amplifying At5g24520(SEQ ID NO: 18) AACTCTAAGGAGCTGCATTTTGTTAGCAAA Forward primer for amplifying At5g07580 (SEQ ID NO: 19)ATGGCGAGTTTTGAGGAAAGC  Reverse primer for amplifying At5g07580(SEQ ID NO: 20) AAATGCATCACAGGAAGATGAAG Forward primer for amplifying At1g74930 (SEQ ID NO: 21)ATGGTGAAGCAAGCGATGAAGG  Reverse primer for amplifying At1g74930(SEQ ID NO: 22) AAAATCCCAAAGAATCAAAGATTC Forward primer for amplifying At5g47390 (SEQ ID NO: 23)GATGACTCGTCGATGTTCTCACTGCAATCA  Reverse primer for amplifying At5g47390(SEQ ID NO: 24) TAAAGCGTGTATCACGCTTTTGATGTCTGA Forward primer for amplifying At5g25190 (SEQ ID NO: 25)ATGGCACGACCACAACAACGC  Reverse primer for amplifying At5g25190(SEQ ID NO: 26) CAGCGTCTGAGTTGGTAAAACAG Forward primer for amplifying At3g61910 (SEQ ID NO: 27)GATGAACATATCAGTAAACGGACAGTCACA  Reverse primer for amplifying At3g61910(SEQ ID NO: 28) TCCACTACCGTTCAACAAGTGGCATGTCGT Preparation of Fusion Genes

Fusion genes that encode fusion proteins of the above transcriptionfactors each comprising a repressor domain sequence added to the Cterminus were prepared. In order to add a polynucleotide encoding arepressor domain sequence to the 3′ terminus of each of the DNAfragments amplified via PCR above, the p35SSXG vector having the SmaIsite and a polynucleotide encoding the repressor domain sequence(GLDLDLELRLGFA) in a site downstream of the CaMV 35S promoter was firstprepared. p35SSXG was cleaved with SmaI and the DNA fragments amplifiedvia PCR above were inserted thereinto. The resulting expression vectorswere designated as p35SSXG (At3g15510), p35SSXG (At5g24520), p35SSXG(At5g07580), p35SSXG (At1g74930), p35SSXG (At5g47390), p35SSXG(At5g25190), and p35SSXG (At3g61910).

Construction of Binary Vectors

A pBCKH binary vector was used in order to transform a plant by theAgrobacterium method. This vector was prepared by incorporating acassette of the Gateway vector conversion system (Invitrogen) into theHindIII site of pBIG (Hygr) (Nucleic Acids Res. 18, 203, 1990). In orderto incorporate the fusion gene into this vector, the vector was mixedwith p35SSXG (At3g25890) or p35SSXG (At1g56650), and a recombinationreaction was carried out using GATEWAY LR clonase (Invitrogen). As aresult, pBCKH-p35SSXG (At3g15510), pBCKH-p35SSXG (At5g24520),pBCKH-p35SSXG (At5g07580), pBCKH-p35SSXG (At1g74930), pBCKH-p35SSXG(At5g47390), pBCKH-p35SSXG (At5g25190), and pBCKH-p35SSXG (At3g61910)were constructed.

Introduction of Binary Vector into Plant

In this example, Arabidopsis thaliana of Brassicaceae (Arabidopsisthaliana, Columbia) was used. Gene introduction was carried out inaccordance with the method described in Bechtold, E., Ellis, J., andPelletier, G., 1993, In Planta Agrobacterium-mediated gene transfer byinfiltration of adult Arabidopsis plants, C. R. Acad. Sci. Paris Sci.Vie, 316, 1194-1199 and Zyprian E., Kado C. L., Agrobacterium-mediatedplant transformation by novel mini-T vectors in conjunction with ahigh-copy vir region helper plasmid, Plant Molecular Biology, 1990, 15(2), 245-256. Plants were infected via soaking in the Agrobacteriumsolution without depressurization. Specifically, the binary vectorsconstructed above were introduced into soil bacteria (i.e., theAgrobacterium tumefaciens strain GV3101 (C58C1Rifr) pMP90 (Gmr)) (konczand Schell, 1986) via electroporation. The introduced bacteria werecultured in 1 liter of YEP medium containing antibiotics (50 μg/ml ofkanamycin (Km), 25 μg/ml of gentamicin (Gm), and 50 μg/ml of rifampicin(Rif)) until OD600 reached 1. Subsequently, the bacteria were recoveredfrom the culture solution and suspended in 1 liter of infiltrationmedium (containing 2.2 g of MS salt, 1×B5 vitamins, 50 g of sucrose, 0.5g of MES, 0.044 μM of benzylaminopurine, and 400 μl of Silwet per liter;pH: 5.7).

The Arabidopsis thaliana plant that had been grown for 14 days wassoaked in this solution for 1 minute, the plant was infected, andculture was continued again for fructification. The resulting seeds (T1seeds) were sterilized with a 50% bleach/0.02% Triton X-100 solution for7 minutes, the seeds were rinsed three times with sterilized water, andthe seeds were sowed on the sterilized hygromycin selection medium (4.3g/l MS salts, 0.5% sucrose, 0.5 g/l MES (pH 5.7), 0.8% agar, 30 mg/lhygromycin, and 250 mg/l vancomycin). Ten transformed strains that hadgrown on the hygromycin plate (T1 plants) were selected per modifiedtranscription gene and transferred to a pot (diameter: 50 mm) containingvermiculite composite soils. The transformants were cultivated at 22° C.for 16 hours in the light and 8 hours in the dark at an opticalintensity of about 60 to 80 μE/cm² to obtain seeds (T2 seeds).

<Analysis of T2 Seeds>

Quantitative analysis of fat and oil components in the resulting T2seeds was carried out using MARAN-23 (Resonance Instruments Ltd.,UK)^(H)-NMR and the RI-NMR Ver. 2.0 analysis software. With the use ofsuch apparatuses, 2 to 10 mg of T2 seeds were measured. A calibrationcurve was prepared using olive oil as the fat and oil reference materialand the fat and oil content in the seeds (% by weight) was determined.

Single seed weight was measured by weighing about 1 mg of T2 seeds,spreading the T2 seeds on a glass petri dish, scanning the image ofseeds using Pictrostat (Fujifilm), gray-scale processing the image usingPhotoshop image-editing software, analyzing the gray-scale image usingScion Image image-analyzing software, and determining the number ofseeds. The total seed weight was divided by the number of seeds, and theseed weight per grain was determined. The fat and oil components ofwild-type Arabidopsis thaliana were similarly quantified. The resultsare summarized in Table 2.

TABLE 2 Fat and oil content Percentage of Single seed weight increase infat Percentage Fat and oil amount per grain Name of introduced and oilof increase Amount of fat and Percentage of gene Content (%) contentWeight (μg) in weight oil (μg/grain) increase WT 34.3% 19.8 6.8At3g15510-SRDX 42.4% 23.7% 20.4 3.1% 8.64 26.9% At5g24510-SRDX 42.3%23.4% 19.8 0.3% 8.39 23.2% At5g07580-SRDX 42.2% 23.2% 18.2 −7.8% 7.6913.0% At1g74930-SRDX 42.0% 22.5% 18.8 −4.8% 7.90 16.0% At5g47390-SRDX41.2% 20.2% 27.3 38.2% 11.25 65.2% At5g25190-SRDX 41.2% 20.1% 25.3 28.3%10.44 53.3% At3g61910-SRDX 41.2% 20.1% 17.6 −10.7% 7.26 6.6%

As is apparent from Table 2, the percentage of increase in the fat andoil amount per grain is significantly increased in all the plantsprepared in the examples, compared with wild-type plants. The plantsinto which the transcription factor At5g47390 with suppressedtranscription accelerating activity had been introduced and the plantsinto which the transcription factor At5g25190 with suppressedtranscription accelerating activity had been introduced exhibitedexcellent percentages of increase in the fat and oil amount per grain.

As is apparent from Table 2, the fat and oil content in seeds of controlwild-type plants into which no gene had been introduced was 34.3% andthe single seed weight thereof was 19.8 μg. In contrast, the fat and oilcontent in seeds of all the plants prepared in the examples wasincreased by 20% or more from that in wild-type plants. Three strains(i.e., At3g15510-SRDX, At5g24520-SRDX, and At5g07580-SRDX) exhibited anincrease in the fat and oil content of 23% or more.

The above results demonstrate that the plants into which thetranscription factors with suppressed expression accelerating activityhad been introduced exhibit the excellent fat and oil content per grainand such plants are thus very effective for fat and oil production.

<Analysis of T3 Seeds>

In order to analyze T3 seeds, the T2 plants prepared as above werecultivated via two separate experiments. Because of differentillumination conditions resulting from the different positions ofcultivation trays, test plants and control plants were simultaneouslycultivated in the same cultivation tray, and the results were compared.

Experiment 1)

After the T2 seeds were sterilized with a 50% bleach/0.02% Triton X-100solution for 7 minutes, the seeds were rinsed three times withsterilized water, and the seeds were sowed on the sterilized seedingmedium (4.3 g/l MS salts, 0.5% sucrose (pH 5.7), 0.8% agar, and 10 mg/lhygromycin). Three weeks after seeding, 4 each transformed plants (T1plants) into which the modified transcriptional genes had beenintroduced were transferred to a pot (diameter: 50 mm) containingvermiculite composite soils. As controls, 2 non-recombinant Arabidopsisthaliana plants were transferred. Each strain of the plants wasseparately introduced into cultivation trays and cultivated at 22° C.for 16 hours in the light and 8 hours in the dark at an opticalintensity of about 30 to 45 μE/cm², and, 4 weeks thereafter, the plantswere subjected to thinning out while leaving 4 recombinant plants and 3non-recombinant plants behind. The plants were cultivated for anadditional 7 weeks until 11 weeks after the transfer.

Experiment 2)

Seeds were sterilized, sowed on plates, and grown in the same manner asin Experiment 1), and 6 each transformed plants (T1 plants) into whichthe modified transcriptional genes had been introduced were transferredto a pot (diameter: 50 mm) containing vermiculite composite soils.Cultivation was carried out in the same manner as in Experiment 1), andthe plants were cultivated until 11 weeks after the transfer.

Measurement and Analysis)

The aerial parts of the plants were introduced into a paper bag anddried at 22° C. and humidity of 60% for 2 weeks. Thereafter, totalbiomass amount and seed yield were weighed using an electronic balance.Quantitative analysis of fat and oil was carried out by the methoddescribed above.

FIG. 1 shows the results of measuring the fat and oil content of T3seeds. The fat and oil contents in the seeds obtained from the controlwild type (WT) plant was not consistent with the results of measurementof T2 seeds, which had been cultivated under different conditions. Thetest strains into which the transcription factors At5g24520-SRDX,At5g07580-SRDX, and At5g61910-SRDX with suppressed expressionaccelerating activity had been introduced exhibited higher fat and oilcontents in seeds than the control strains. FIG. 2 shows the seedyields. The strain into which At5g07580-SRDX had been introducedexhibited the seed yield increased by about 42% from that of the controlstrain. The amount of fat and oil production per plant was calculatedbased on the product of the seed yield and the fat and oil content, andthe results are shown in FIG. 3. The strain into which At5g07580-SRDXhad been introduced exhibited a significantly higher fat and oil contentper plant than the control WT strain. FIG. 4 shows the results ofmeasuring the total biomass amount of the aerial parts including seeds.The strains into which the transcription factors At5g07580-SRDX andAt5g25190-SRDX with suppressed transcription accelerating activity hadbeen introduced exhibited a significantly higher total biomass amountthan control WT strains.

Table 3 shows the percentage of increase/decrease in fat and oilcontent, seed yield, fat and oil amount per plant, and biomass amount ofrecombinant test strains into which the transcription factor genes withthe regulated transcription accelerating activity had been introduced bydesignating the values of the control strains as 100%.

TABLE 3 Percentage of increase/decrease (relative to the control = 100%)Fat and oil Seed Fat and oil Biomass Tested strain content yield amountper plant amount At3g15510-SRDX 100% 43% 49% — At5g24520-SRDX 109% 113% 113%  104% At5g07580-SRDX 114% 142%  151%  125% At1g74930-SRDX 105%110%  116%  104% At5g47390-SRDX  67% 60% 80%  96% At5g25190-SRDX  83%94% 93% 113% At5g61910-SRDX 109% 80% 78%  74%

When the T2 generation is compared with the T3 generation, the aboveresults occasionally show differences in fat and oil content per plant,seed yield, fat and oil content, and the amount of biomass. Because ofthe application of Mendel's law for the case of the difference betweenthe T2 generation and the T3 generation, the T2 generation and the T3generation do not always have the same genotype. Since mRNA may suppressgene expression as is known in the case of the RNAi technique, also,differences occur between the T2 generation and the T3 generation. Theplants into which any of the transcription factor At3g15510, At5g24520,At5g07580, At1g74930, At5g47390, At5g25190, or At3g61910 with suppressedexpression accelerating activity had been introduced can be evaluated asexhibiting excellent effects in terms of increased biomass amount,increased seed yield, and increased fat and oil yield.

Example 2

In Example 2, a fusion protein of the Arabidopsis thaliana transcriptionfactor At5g07580 to which a repressor domain sequence had been added wasexpressed in plants as in the case of Example 1, and the fat and oilcontent in seeds obtained from rice of graminaceous monocotyledonousplants (Olyza sative Nipponbare) was measured.

Amplification of Transcription Factor Gene, Preparation of Fusion Gene,and Construction of Binary Vector

Amplification of the transcription factor gene, preparation of thefusion gene, and construction of the binary vector were carried out inthe same manner as in Example 1.

Introduction of Binary Vector into Plant

A binary vector was introduced into rice plants (Nipponbare) usingAgrobacterium carrying the binary vector in accordance with the methoddescribed in JP Patent No. 3141084 to obtain calluses.

The calluses into which the gene had been introduced were subjected toselection with hygromycin at 50 ppm for a month, and calluses exhibitingdrug resistance were obtained. DNA was prepared from the obtainedcalluses in accordance with a conventional technique. The At5g07580fusion gene was confirmed via PCR using the prepared DNA as a template.The calluses having drug-resistance phenotypes and containing theAt5g07580 fusion gene were transferred to a redifferentiation medium(described in JP Patent No. 3141084) to induce redifferentiation, andthe resultant was then transferred to a hormone-free MS medium(described in JP Patent No. 3141084) to obtain transformed plants.

The transformed plants were grown for 16 hours in the light (photonamount: 135 μE/cm²; temperature: 30° C.) and for 8 hours in the dark(temperature: 25° C.) for 100 days. Thereafter, the plants were furthergrown for 12 hours in the light (photon amount: 135 μE/cm²; temperature:30° C.) and for 12 hours in the dark (temperature: 25° C.), and thefructified seeds (T1 seeds) were recovered.

Analysis of T1 Seeds

Fat and oil in the resulting rice T1 seeds was quantitatively analyzedin the same manner as in <Analysis of T2 seeds> in Example 1. Since therice seed weight is about 20 mg per brown rice grain, the fat and oilcontent in a grain was quantified with good reproducibility. The resultsare shown in Table 4. Brown rice is a seed containing a pericarp, a seedcoat, an albumen, and an aleurone layer, and caryopsis is a so-calledhull.

TABLE 4 Fat and oil content Single seed weight Fat and oil amount pergrain Name of Name of Content Percentage of Weight Percentage of Fat andoil Percentage of introduced gene Strain Tissue (%) increase (mg)increase amount (mg) increase WT (average of 5 grains) Brown rice 2.17 —20.90 — 0.454 — At5g07580-SRDX CR035-10-5 Brown rice 1.93 −11.1% 24.0114.9% 0.463 2.2% At5g07580-SRDX CR035-15-2 Brown rice 3.10 42.9% 17.92−14.3% 0.556 22.5% At5g07580-SRDX CR035-18-2 Brown rice 3.14 44.7% 16.37−21.7% 0.514 13.3% WT (average of 5 grains) Caryopsis 5.91 — 3.99 —0.236 — At5g07580-SRDX CR035-10-5 Caryopsis 5.88 −3.9% 4.65 16.5% 0.26412.0% At5g07580-SRDX CR035-12-1 Caryopsis 7.63 29.1% 4.56 14.3% 0.34847.5% At5g07580-SRDX CR035-20-3 Caryopsis 10.35 75.1% 2.76 −30.8% 0.26621.1%

As is apparent from Table 4, graminaceous monocotyledonous plants intowhich the transcription factor At5g07580 with suppressed expressionaccelerating activity had been introduced exhibited a fat and oilcontent much higher than that of wild-type plants. Such transformedplants exhibited the excellent percentages of increase in fat and oilcontent per grain of 44.7% in brown rice and 75.1% in caryopsis, theexcellent percentages of increase in the seed weight of 14.9% in brownrice and 16.5% in caryopsis, and the excellent percentages of increasein the fat and oil amount per seed grain of 22.5% in brown rice and47.5% in caryopsis.

All publications, patents, and patent applications cited herein areincorporated herein by reference in their entirety.

The invention claimed is:
 1. A plant which expresses a chimeric protein,said chimeric protein comprising a transcription factor fused to arepressor domain sequence that converts said transcription factor into atranscription repressor, wherein said transcription factor is a proteincomprising the amino acid sequence of SEQ ID NO: 12, or a proteincomprising the amino acid sequence of SEQ ID NO: 12 but in which 1-20amino acids have been deleted, substituted, added or inserted, andwherein the seeds of said plant exhibit an increase in weight, anincrease in amount of fat and oil, or an increase in fat and oilcontent, in comparison to a wild-type plant not expressing said chimericprotein.
 2. The plant according to claim 1, wherein the repressor domainsequence comprises an amino acid sequence selected from the groupconsisting of (1) to (8): (1) X1-Leu-Asp-Leu-X2-Leu-X3 (SEQ ID NO: 29with deletion of 0-10 residues from the N-terminus) wherein X1represents 0 to 10 amino acid residues; X2 represents Asn or Glu; and X3represents at least 6 amino acid residues; (2) Y1-Phe-Asp-Leu-Asn-Y2-Y3(SEQ ID NO: 30 with deletion of 0-10 residues from the N-terminus)wherein Y1 represents 0 to 10 amino acid residues; Y2 represents Phe orIle; and Y3 represents at least 6 amino acid residues; (3)Z1-Asp-Leu-Z2-Leu-Arg-Leu-Z3 (SEQ ID NO: 31 with deletion of 0-10residues from the C-terminus and deletion of 0-2 residues from theN-terminus) wherein Z1 represents Leu, Asp-Leu, or Leu-Asp-Leu; Z2represents Glu, Gln, or Asp; and Z3 represents 0 to 10 amino acidresidues; (4) (SEQ ID NO: 32) Asp-Leu-Z4-Leu-Arg-Leu 

wherein Z4 represents Glu, Gln, or Asp; (5) (SEQ ID NO: 33)α1-Leu-β1-Leu-γ1-Leu; (6) (SEQ ID NO: 34) α1-Leu-β1-Leu-γ2-Leu; (7)(SEQ ID NO: 35) α1-Leu-β2-Leu-Arg-Leu; and (8) (SEQ ID NO: 36)α2-Leu-β1-Leu-Arg-Leu;

wherein, in formulae (5) to (8), α1 represents Asp, Asn, Glu, Gln, Thr,or Ser; α2 represents Asn, Glu, Gln, Thr, or Ser; β1 represents Asp,Gln, Asn, Arg, Glu, Thr, Ser, or His; β2 represents Asn, Arg, Thr, Ser,or His; γ1 represents Arg, Gln, Asn, Thr, Ser, His, Lys, or Asp; and γ2represents Gln, Asn, Thr, Ser, His, Lys, or Asp.
 3. A method forproducing a plant in which the seeds of said plant exhibit an increasein weight, an increase in amount of fat and oil, or an increase in fatand oil content, said method comprising the step of expressing achimeric protein in a plant, said chimeric protein comprising atranscription factor fused to a repressor domain sequence that convertssaid transcription factor into a transcription repressor, wherein saidtranscription factor is a protein comprising the amino acid sequence ofSEQ ID NO: 12, or a protein comprising the amino acid sequence of SEQ IDNO: 12 but in which 1-20 amino acids have been deleted, substituted,added or inserted, and wherein the seeds of said plant exhibit anincrease in weight, an increase in amount of fat and oil, or an increasein fat and oil content, in comparison to a wild-type plant notexpressing said chimeric protein.
 4. The method for producing a plantaccording to claim 3, wherein the repressor domain sequence comprises anamino acid sequence selected from the group consisting of (1) to (8):(1) X1-Leu-Asp-Leu-X2-Leu-X3 (SEQ ID NO: 29 with deletion of 0-10residues from the N-terminus) wherein X1 represents 0 to 10 amino acidresidues; X2 represents Asn or Glu; and X3 represents at least 6 aminoacid residues; (2) Y1-Phe-Asp-Leu-Asn-Y2-Y3 (SEQ ID NO: 30 with deletionof 0-10 residues from the N-terminus) wherein Y1 represents 0 to 10amino acid residues; Y2 represents Phe or Ile; and Y3 represents atleast 6 amino acid residues; (3) Z1-Asp-Leu-Z2-Leu-Arg-Leu-Z3 (SEQ IDNO: 31 with deletion of 0-10 residues from the C-terminus and deletionof 0-2 residues from the N-terminus) wherein Z1 represents Leu, Asp-Leu,or Leu-Asp-Leu; Z2 represents Glu, Gln, or Asp; and Z3 represents 0 to10 amino acid residues; (4) (SEQ ID NO: 32) Asp-Leu-Z4-Leu-Arg-Leu

wherein Z4 represents Glu, Gln, or Asp; (5) (SEQ ID NO: 33)α1-Leu-β1-Leu-γ1-Leu; (6) (SEQ ID NO: 34) α1-Leu-β1-Leu-γ2-Leu; (7)(SEQ ID NO: 35) α1-Leu-β2-Leu-Arg-Leu; and (8) (SEQ ID NO: 36)α2-Leu-β1-Leu-Arg-Leu,

wherein, in formulae (5) to (8), α1 represents Asp, Asn, Glu, Gln, Thr,or Ser; α2 represents Asn, Glu, Gln, Thr, or Ser; β1 represents Asp,Gln, Asn, Arg, Glu, Thr, Ser, or His; β2 represents Asn, Arg, Thr, Ser,or His; γ1 represents Arg, Gln, Asn, Thr, Ser, His, Lys, or Asp; and γ2represents Gln, Asn, Thr, Ser, His, Lys, or Asp.
 5. A method forisolating fat and oil from a plant, comprising separating and recoveringfat and oil from the seeds of a plant which expresses a chimericprotein, wherein said chimeric protein comprises a transcription factorfused to a repressor domain sequence that converts said transcriptionfactor into a transcription repressor, wherein said transcription factoris a protein comprising the amino acid sequence of SEQ ID NO: 12, or aprotein comprising the amino acid sequence of SEQ ID NO: 12 but in which1-20 amino acids have been deleted, substituted, added or inserted, andwherein the seeds of said plant exhibit an increase in weight, anincrease in amount of fat and oil, or an increase in fat and oilcontent, in comparison to a wild-type plant not expressing said chimericprotein.
 6. A chimeric protein comprising a transcription factor fusedto a repressor domain sequence that converts said transcription factorinto a transcription repressor, wherein said transcription factor is aprotein comprising the amino acid sequence of SEQ ID NO: 12, or aprotein comprising the amino acid sequence of SEQ ID NO: 12 but in which1-20 amino acids have been deleted, substituted, added or inserted, andwherein when said chimeric protein is expressed in a plant, said plantproduces seeds that exhibit an increase in weight, an increase in amountof fat and oil, or an increase in fat and oil content, in comparison toa wild-type plant not expressing said chimeric protein.
 7. Apolynucleotide encoding the chimeric protein according to claim
 6. 8. Arecombinant expression vector comprising the polynucleotide according toclaim 7 and a promoter.
 9. A kit comprising the expression vectoraccording to claim
 8. 10. The kit according to claim 9, wherein said kitfurther comprises reagents for introducing the recombinant expressionvector into a plant cell.